Crystal Structures of Multicopper Oxidase Cueo G304K Mutant

www.nature.com/scientificreports OPEN Crystal structures of multicopper oxidase CueO G304K mutant: structural basis of the increased Received: 17 November 2017 Accepted: 5 September 2018 laccase activity Published: xx xx xxxx Hanqian Wang1, Xiaoqing Liu2, Jintong Zhao2, Qingxia Yue2, Yuhua Yan1,3, Zengqiang Gao4, Yuhui Dong1,4, Zhiyong Zhang5, Yunliu Fan2, Jian Tian2, Ningfeng Wu2 & Yong Gong1 The multicopper oxidase CueO is involved in copper homeostasis and copper (Cu) tolerance in Escherichia coli. The laccase activity of CueO G304K mutant is higher than wild-type CueO. To explain this increase in activity, we solved the crystal structure of G304K mutant at 1.49 Å. Compared with wild- type CueO, the G304K mutant showed dramatic conformational changes in methionine-rich helix and the relative regulatory loop (R-loop). We further solved the structure of Cu-soaked enzyme, and found that the addition of Cu ions induced further conformational changes in the R-loop and methionine-rich helix as a result of the new Cu-binding sites on the enzyme’s surface. We propose a mechanism for the enhanced laccase activity of the G304K mutant, where movements of the R-loop combined with the changes of the methionine-rich region uncover the T1 Cu site allowing greater access of the substrate. Two of the G304K double mutants showed the enhanced or decreased laccase activity, providing further evidence for the interaction between the R-loop and the methionine-rich region. The cuprous oxidase activity of these mutants was about 20% that of wild-type CueO. These structural features of the G304K mutant provide clues for designing specifc substrate-binding mutants in the biotechnological applications. Multicopper oxidases (MCOs) are a large, widely distributed and diverse family of enzymes with various functions ranging from copper (Cu) and iron metabolism to polyphenol oxidation1. One important feature of MCOs is that a minimum of four Cu ions are arranged at two sites: the mononuclear type 1 Cu centre (T1), and the tri- nuclear Cu cluster (TNC) consisting of a type 2 Cu centre (T2) and a dinuclear type 3 centre (T3)1,2. Four single electron-transfer reactions from the T1 site to the TNC cluster are coupled to the oxidation of various substrates. Electrons are passed to dioxygen bound to the TNC to generate two water molecules. Laccases are the largest subfamily of MCOs and widely distributed in diverse organisms. Tey have multi- ple functions, including lignin biosynthesis and wound healing in plants; lignin degradation, pigmentation, and pathogenesis in fungi; and pigment production in the endospore coat in some bacteria such as Bacillus subtilis3. Laccases can catalyse the oxidation of a broad spectrum of organic substrates such as polyphenols, diamines, and some inorganic compounds4. Tese enzymes have been received increasing attention because of their practical applications in the textile, food, and wood-processing industries and their possible uses in bioremediation5. CueO, an MCOs from Escherichia coli (E. coli), together with CopA, a P-type ATPase, comprises a copper efux (cue) system that resists copper stress under aerobic conditions6,7. Expression of both enzymes is stim- ulated by exogenous Cu ions via the cytosolic metalloregulatory protein CueR8,9. Previous analysis of the crystal structure of E. coli CueO has highlighted that it adopts a canonical architecture consisting of three similar 1Multi-discipline Center, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China. 2Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. 3Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui, 230601, China. 4Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China. 5CAS Key Laboratory for Biomedical Efects of Nanomaterials and Nanosafety, CAS Key Laboratory of Nuclear Radiation and Nuclear Energy Technology, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China. Hanqian Wang and Xiaoqing Liu contributed equally. Correspondence and requests for materials should be addressed to J.T. (email: [email protected]) or Y.G. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:14252 | DOI:10.1038/s41598-018-32446-7 1 www.nature.com/scientificreports/ cupredoxin-like domains linked by peptides. Tis architecture is shared by the other known laccase and ascorbate oxidase. Within domain III, a unique 42-residue methionine-rich region made up mainly of methionine-rich helix (thereafer, MR helix) may hinder the access of bulky organic substrates to the T1 Cu site10. Tis region near the T1 Cu position was later found to coordinate a labile ffh Cu atom, a novel feature of CueOs that difers from other MCOs11. Like other common laccases, CueO exhibits phenol oxidase activity with a broad range of substrates. However, this enzyme possesses an unique cuprous oxidase activity in vitro6,12. CueO can catalyze the oxidation of cuprous ion (Cu(I)) to the less toxic cupric ion (Cu(II)) in vivo12. Recent structural and functional studies of engineered E. coli CueO have showed that removal of the methionine-rich helical region signifcantly decreased cuprous oxidase activity of the enzyme, but increased its phenol oxidase activity. Tese fndings implied that the MR helix region confers the specifc cuprous oxidase of CueO13. Djoko et al. provided compelling evidence that the higher afnity of the methionine-rich region for Cu (I) over Cu (II) explains why CueO functions solely as a cuprous oxidase but not phenol oxidase in vivo14. Recent analyses of the crystal structures of CueO bound to Cu (I) provided evidence that the methionine-rich region binds and oxides Cu (I)15. Very recently, we characterized the E. coli CueO G304K mutant (hereafer, G304K) from the metagenome of the sludge in a chemical plant, and found that catalytic efciency of the mutant towards an organic substrate in the presence of excess Cu (II) was 2.7-folds higher than that of the wild-type enzyme16. In the present work, we report the crystal structure of G304K at a resolution of 1.49 Å. We also report the structure of G304K in the presence of excess Cu ions at 2.20 Å. Compared with the wild-type enzyme, G304K showed dramatically conformational changes, with the excess Cu ions inducing the further conformation changes. Combined with the structural information, functional assays highlighted that the regulatory loop (hereafer, R-loop) may modulate the methionine-rich region to regulate the activity of the enzyme. Results Overall structure of G304K in the presence of excess Cu ions. Recently, we characterized G304K, which shows markedly increased the laccase activity compared with that of the wild-type enzyme16. To fnd out the reason for the increase in laccase activity, we determined the crystal structure of G304K at a resolution of 1.49 Å (Table 1). As the maximum laccase activity of CueO is achieved only in the presence of excess Cu ions10, we soaked G304K crystals in Cu-containing solution and determined the complex structure at a resolution of 2.20 Å (Table 1). Te resolved G304K structure is essentially identical to that of wild-type protein10 (PDB entry 1KV7). Similar to most of the reported crystal structures of CueO, the loop (residues 370–398) near T1 Cu atom is dis- ordered in the G304K structure, suggesting that it is fexible (Fig. 1A). One of the most obvious features is the presence of several new Cu-binding sites only found on the surface of G304K structure afer soaked with Cu ions. Te presence of these extra Cu-binding sites was confrmed by the analysis of the anomalous diference Fourier map (Figs 1 and S1A,B). Among these Cu-binding sites, Cu5 and Cu6 were located around the MR helix (residues 357–369). Cu5 was coordinated by His314, located on the R-loop (residues 299–314), and two water moleculars (Fig. 1B). Cu6 was coordinated by His145, His405, and a water molecular (Fig. 1C). Tere were two other Cu ions found in the Cu-soaked G304K structure: Cu7 and Cu8. Cu7 was coordinated by His495, Glu110, and also found in Δα5–7 CueO structure13 (PDB entry 2YXW); whereas Cu8 was linked with His488, Asp132, and a water molecular (Fig. S1A,B). Tis was identical to the coordination environment in the wild-type protein structure11 (PDB entry 1N68), except that the additional water molecular served as a ligand to Cu8 in the G304K crystal structure. In the G304K crystal structure, although the T1 and T3 Cu atoms are fully occupied, T2 Cu atom is depleted. Partial depletion of the four catalytic Cu atoms, especially the T2 Cu atom, was found in crystal structures of some CueO and other MCOs17–20. Analyses of X-ray data collection showed that X-ray radiation can lead to decrease in metal occupancy, even depletion21–24. Notably, the estimated absorbed dose during data collection of G304K crystal structure was 8.9 MGy using RADDOSE-3D25. For CueO, the removal of the T2 Cu atom, and even the loss of all four Cu atoms in the apo-CueO (PDB entry 3NSF), did not afect the overall fold15,20. We tried to fnd a possible Cu-binding site (substrate Cu (sCu)11, frst found in the PDB entry 1N68) located on the MR helix, like the one that exists in the wild-type enzyme structure. When we examined the mutant protein crystal, we found no evidence of a Cu ion at the corresponding site in the structure. Even with excess Cu ions, no electron density that could be assigned as Cu was present on the MR helix of G304K. To exclude the possibility that the Cu content was afected in the preparation or purifcation process of CueO, we measured the total Cu content in the CueO using the ICP-MS (inductively coupled plasma mass spectrom- etry) method: the total Cu content in the purifed wild-type CueO was 5.4 per protein molecule, and that in the G304K 5.1 per protein molecule (the experimental error in determining the Cu content was ca 10%).

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